The site of macromolecular restriction in the renal glomerulus will be defined by serveral new morphological approaches for electron microscopy. First, the permeability barrier in the mouse glomerulus will be probed with three new series of ultrastructural tracers with molecular weights which span the cut-off size for the filter as defined by serum albumin. These tracers will consist of (a) horseradish peroxidase (HRP) conjugated to IgG immunoglobulin and its Fab and F(ab')2 fragments, (b) synthetic oligomers of HRP, and (c) polyvinylpyrrolidone (PVP) fractions of graded molecular weights. HRP-derived tracers will be visualized histochemically with hydrogen peroxide and 3,3' diaminobenzidine. PVP fractions will be detected by a fixation technique which employs tannic acid. With each series of tracers, the relative barrier functions of the basement membrane (BM) and slit diaphragm (SD) will be ascertained. As a second approach, our study of the glomerular wall in the bullfrog will be continued to provide an example where the BM exhibits an altered ultrastructure which may influence its filtration properties. Horse spleen ferritin and peroxidase conjugates will be used as ultrastructural tracers to assess the permeability of the BM and SD to proteins in this species. Finally, studies of the comparative ultrastructure of the SD in several other animal species will be continued to determine whether the characteristic structure of the SD found in each species can be correlated with the filtration properties of the kidney in that species. BIBLIOGRAPHIC REFERENCES: Schaffner, A., and R. Rodewald. 1975. Glomerular filtration of ferritin in the bullfrog Rana catesbeiana. J. Cell Biol. 67: 384a. Schaffner, A., and R. Rodewald. 1976. A comparative study of filtration slit diaphragm in vertebrates and invertebrates. J. Cell Biol. 70: 330a.